Is there a direct role for erythrocytes in the immune response?

Erythrocytes are highly abundant circulating cells in the vertebrates, which, with the notable exception of mammals, remain nucleated throughout the entire life cycle. The major function associated with these cells is respiratory gas exchange however other functions including interaction with the immune system have been attributed to these cells. Many viral, prokaryotic and eukaryotic pathogens directly target this cell type and across the vertebrate group a significant number of related pathologies have been reported. Across the primary literature mechanisms of interaction, invasion and replication between viruses and erythrocytes have been well described however the functional response of the erythrocyte has been poorly studied. A fragmented series of reports spanning the vertebrates suggests that these cells are capable of functional responses to viral infection. In contrast, in-depth proteomic studies using human erythrocytes have strongly progressed throughout the past decade providing a rich source of information related to protein expression and potential function. Furthermore information at the gene expression level is becoming available. Here we provide a review of erythrocyte-pathogen interactions, erythrocyte functions in immunity and propose in light of recent -omics research that the nucleated erythrocytes may have a direct role in the immune response.

Detection of Bartonella quintana by direct immunofluorescence examination of blood smears of a patient with acute trench fever.

We report a case of Bartonella quintana acute symptomatic infection in a homeless man, presenting as a typical trench fever. B. quintana has been retrieved in erythrocytes in large clusters and in erythroblasts. Direct immunofluorescence of blood smears allows a rapid diagnosis.

Phosphorylation of the SHC proteins on tyrosine correlates with the transformation of fibroblasts and erythroblasts by the v-sea tyrosine kinase.

The S13 avian erythroblastosis viral genome encodes an oncogenic tyrosine kinase, termed env-sea, that is capable of transforming fibroblasts and erythroblasts. Although the tyrosine kinase activity of the env-sea protein has been shown to be necessary for transformation, no substrates for this enzyme have been detected in vivo. Here we demonstrate that the recently described shc proteins are phosphorylated on tyrosine residues in both S13 transformed fibroblasts and erythroblasts. Furthermore, using an S13 temperature sensitive mutant, we show that the phosphorylation of the shc proteins occurs concomitantly with the activation of the tyrosine kinase activity of the env-sea protein. These observations make the phosphorylation of the shc proteins a good candidate for being involved in oncogenic signaling by the env-sea oncoprotein.

Persistent infection of human erythroblastoid cells by poliovirus.

Human erythroblastoid K562 cells have been recently described as a relatively nonpermissive host for poliovirus replication. During investigations of virus-induced cytopathic effects in this cell line, we discovered that poliovirus easily established persistently infected cultures in K562-Mu cells. In these cultures, most cells remained viable, with overall viability continuously maintained between 67 and 92% over 3 months. Infected K562 cells continued to grow, usually without any major periods of crisis in the culture or large diminutions in cell growth rate. K562-Mu cells produced a slower onset of virus production than observed in HeLa cells, and virus titers in culture supernates rapidly stabilized at levels between 10(5) and 10(6) PFU/ml. In infectious center or limiting dilution assays, only about 10% of K562 cells produced infectious virus after 2 days. However, when assays were extended to 3 to 5 days, most K562 cells in the culture scored as infectious centers, suggesting productive infection of all cells in the culture with delayed kinetics of virus production. Cultures of infected K562 cells could not be cured of virus by prolonged incubation with high-titer neutralizing antibody. Pulse-label SDS-PAGE analysis of infected cultures detected moderate levels of virus protein synthesis which peaked at 9-12 hr postinfection; however, little or no shutoff of host protein synthesis was observed at any time point during infection. Immunoblot analysis with antisera to the p220 subunit of eIF-4F demonstrated extensive but incomplete cleavage of p220 in infected K562 cells at times which correlated with peak viral protein synthesis. Taken together, the results demonstrate a persistent infection in which host cell shutoff does not occur despite significant viral protein synthesis and extensive early degradation of p220.

Transcriptional repression of band 3 and CAII in v-erbA transformed erythroblasts accounts for an important part of the leukaemic phenotype.

The v-erbA oncogene confers two prominent properties on transformed erythroblasts: a block of spontaneous differentiation and tolerance to wide variations in the pH or ionic strength of culture medium. V-erbA acts as a constitutive repressor of erythrocyte-specific gene transcription, arresting the expression of at least three different erythroid genes: the erythrocyte anion transporter (band 3), carbonic anhydrase II (CAII) and delta-aminolevulinate synthase (ALA-S). To test whether or not the v-erbA induced repression of these genes is causally related to the v-erbA induced leukaemic phenotype, we have reintroduced the genes for band 3 or CAII into transformed erythroblasts via retrovirus vectors. We show here that such erythroblasts, expressing v-erbA, require the same narrow range of medium pH and ion concentration for growth as do transformed erythroblasts lacking v-erbA, i.e. the v-erbA induced tolerance to pH variation was abrogated. The v-erbA induced differentiation block, however, remained unaffected by the re-expression of band 3 and was only slightly affected by the re-expression of CAII. Our experiments show that the two v-erbA-related 'erythroblast transformation parameters' are separable: suppression of band 3 and CAII accounts for one parameter (pH/ion tolerance), while the second parameter (differentiation block) must involve v-erbA regulation of a different set of target genes.

[Blood picture findings in children with Parvovirus B19 infections (fifth disease/erythema infectiosum)]. / Blutbildbefunde bei Kindern mit Parvovirus B19-Infektion (Ringelröteln/Erythema infectiosum).

The human parvovirus B19 provokes erythema infectiosum ("e.i."); moreover there is a wide range of diseases due to parvovirus B19 without exanthema/rash. The erythropoietic blast cells of the bone marrow seems to be the main target cells for this virus. Therefore in cases of prenatal infection the consequences are extremely similar to fetal erythroblastosis ("non-immunological" fetal hydrops). In postnatal life the parvovirus B19 infection causes hyporegenerative phases of the erythropoiesis with anaemia after 3-4 weeks. We studied the white blood cell count (WBC), erythrocytes and thrombocytes in children suffering from (serologically well documented) parvovirus B19 infection with exanthem/"e.i." (group 1; n = 23), without exanthem (group 2; n = 46) and with unknown febrile exanthematous rashes (group 3; n = 76). We did not find any characteristic data in the WBC for a diagnosis of parvovirus B19 infection. However we have for the first time documented a significant thrombocytopenia in "e.i." (group 1) not found in group 2. The thrombocytopenia appears earlier than the anaemia, because the lifespan of thrombocytes is considerably shorter than that of erythrocytes. These data suggest that parvovirus B19 attacks not only "erythropoietic" blast cells but also immature bone marrow cells, which are later responsible for the thrombocytopoiesis.

[Molecular biological demonstration of parvovirus infection in fetal tissue]. / Molekularbiologischer Nachweis einer Parvovirusinfektion in fetalem Gewebe.

We report on the morphological findings in 16 fetuses with serologically confirmed maternal parvovirus B19 infection. Typical routine morphological findings of the hydropic fetuses were the presence of abnormal erythroblasts with typical nuclear inclusions. These infected cells positively stained immunohistochemically with antibodies against a recombinant virus protein, as well as they ultrastructurally contained virus particles. Using in-situ hybridization techniques, we were able to demonstrate the presence of parvovirus B19 genome in the infected cells, while no other cell type was shown to contain virus genome. According to our results the differential diagnosis of fetal parvovirus B19 infection should be considered in each case with hydrops fetalis of unknown origin. The careful routine microscopic examination of fetal tissue may provide evidence for parvovirus infection which should be confirmed by in-situ hybridization analysis.

[Oncogenes and the origin of leukemia. Acute avian leukemia viruses]. / Onkogene und Leukämieentstehung. Untersuchungen an akuten Hühner-Leukämieviren.

Oncogenes have been intimately associated with the genesis of human neoplasms. A particularly useful system to study the mechanism of tumorigenesis is a small group of avian retroviruses that carry two oncogenes. These viruses causes acute leukemias and can transform hematopoietic cells in vitro. The mechanisms by which viral oncogenes affect the growth control and differentiation of their target cells is now understood in fair detail for two of these virus strains. In the avian erythroblastosis virus AEV, the v-erbB oncogene deregulates the growth control of erythroid precursors, while verbA blocks their terminal differentiation into erythrocytes. Based on the findings that v-erbB oncogene corresponds to a mutated growth factor receptor gene and that v-erbA corresponds to a mutated hormone receptor gene, models have been developed that explain the function of these two oncogenes on a molecular basis. The myelomonocytic leukemia virus MH2 acts by a completely different mechanism. In this case, the v-myc oncogene stimulates the proliferation of macrophage-like cells, while the v-mil gene stimulates them to produce their own growth factor, thus leading to autocrine growth. It will be interesting to determine whether the type of mechanisms of oncogene cooperativity elucidated for acute leukemia viruses are also operative during leukemogenesis in humans.

v-erbA specifically suppresses transcription of the avian erythrocyte anion transporter (band 3) gene.

Previous work has established that the v-erbA oncogene inhibits the temperature-induced differentiation of chick erythroblasts transformed with temperature-sensitive oncogene mutants. Here we demonstrate that v-erbA in differentiating erythroblasts specifically arrests expression of the erythrocyte anion transporter (band 3) gene at the transcriptional level. The v-erbA-induced differentiation block can be overcome by inducing cells to differentiate at alkaline pH. Under these conditions, which possibly impair biological activity of v-erbA, the maturing cells now express the anion transporter gene at high levels. However, its transcription is specifically and rapidly suppressed if v-erbA activity is restored by culturing the cells at neutral pH. Similar but less pronounced inhibition of gene expression by v-erbA was observed for the delta-amino-levulinic acid synthase gene. Additional evidence obtained with an inhibitor of band 3 activity suggests that the v-erbA-induced inhibition of band 3 gene expression is at least partly responsible for the differentiation block caused by this oncogene.

Cytoplasmic poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase in AEV-transformed chicken erythroblasts.

Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A+) mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADP-ribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21,000-120,000 range. The Mr 120,000 protein corresponds to automodified poly(ADP-ribose) polymerase. A Mr 21,000 protein acceptor is abundant in PMS and a Mr 34,000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repression-derepression of mRNA is discussed.

v-myb does not prevent the expression of c-myb in avian erythroblasts.

The v-myb oncogene of avian myeloblastosis virus transforms myeloid cells exclusively, both in vivo and in vitro. The c-myb proto-oncogene from which v-myb arose is expressed at relatively high levels in immature hematopoietic cells of the lymphoid, erythroid, and myeloid lineages but not in myeloblasts transformed by v-myb. This finding suggested that the nuclear v-myb gene product p48v-myb might act directly to inhibit the normal expression of the c-myb gene. I have therefore used a selectable avian retroviral vector to express p48v-myb in avian erythroblasts which normally express high levels of the c-myb gene product p75c-myb. The results demonstrate that p48v-myb and p75c-myb can be coexpressed in the nuclei of cloned cells. Therefore, p48v-myb does not invariably prevent the expression of p75c-myb.

Retrovirus-induced feline pure red cell aplasia. Hematopoietic progenitors are infected with feline leukemia virus and erythroid burst-forming cells are uniquely sensitive to heterologous complement.

Feline leukemia virus subgroup C/Sarma (FeLV-C) induces pure red cell aplasia (PRCA) in cats. Just before the onset of anemia, erythroid colony-forming cells (CFU-E) become undetectable in marrow culture, yet normal frequencies of erythroid burst-forming cells (BFU-E)- and granulocyte-macrophage colony-forming cells (CFU-GM) persist. To determine if erythroid progenitors were uniquely infected with retrovirus, marrow mononuclear cells from cats viremic with FeLV-C were labeled with monoclonal antibodies to gp70 and then analyzed with a fluorescence-activated cell sorter. Both erythroid and granulocyte-macrophage progenitors were among cells sorting positively, suggesting that infection of BFU-E alone did not result in PRCA. The results were confirmed by complement (C') lysis studies using baby rabbit or guinea pig sera as sources of C'. These studies also suggested that BFU-E from cats with PRCA were unusually sensitive to C' alone, without the addition of antibody. In further studies, we demonstrated that C' activation was via the classical pathway and that C' sensitivity was unique to BFU-E and not a property of CFU-E, CFU-GM, or progenitors that were capable of giving rise to BFU-E in suspension culture. As BFU-E from cats viremic with FeLV-A/Glasgow-1 or the Rickard strain of feline leukemia virus were not sensitive to C', this finding may relate to the pathogenesis of feline PRCA. We hypothesize that, in cats viremic with FeLV-C, the abnormal C' sensitivity of BFU-E leads to the absence of CFU-E and anemia.

Variation of erythroid and myeloid precursors in the marrow and peripheral blood of volunteer subjects infected with human parvovirus (B19).

Infection of normal individuals with human parvovirus (B19) results in a mild disease (erythema infectiosum) but gives rise to aplastic crises in patients with chronic hemolytic anemias. The effects of this disease on hemopoiesis were investigated following intranasal inoculation of the virus into three volunteers. A typical disease ensued with a viremia peaking at 9 d. Marrow morphology 6 d after inoculation appeared normal but at 10 d there was a severe loss of erythroid precursors followed by a 1-2-g drop in hemoglobin, and an increase in serum immunoreactive erythropoietin. Erythroid burst-forming units (BFU-E) from the peripheral blood were considerably reduced, starting at the time of viremia and persisting for 4-8 d depending on the individual. Granulocyte-macrophage colony-forming units (CFU-GM) were also affected but the loss started 2 d later. Both CFU-GM and BFU-E showed a sharp overshoot at recovery. In the marrow, BFU-E and CFU-E were reduced at 6 and 10 d in the individual having the longest period of peripheral progenitor loss. In contrast, there was an increase in BFU-E and CFU-E in the subject with least change in peripheral progenitors. In the third subject, with an intermediate picture, there was a loss at 6 d but an increase at 10 d of erythroid progenitors. It is suggested that the architecture of the marrow might partially isolate progenitors from high titers of virus in the serum and individual variation in this respect might give the results observed.

Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing.

The replication of type 1 poliovirus in 13 established human blood cell lines differing in the differentiation stage and cell lineage was investigated. Three T (CCRF-CEM, CCRF-HSB-2, and Molt-3) and three B (Raji, CCRF-SB, and RPMI 8226) cell lines showed no cytopathic effects (CPE) or virus production. CPE associated with virus production were detected in the other seven cell lines: HL-60, ML-1, and KG-1 (granulocytic lineage), U-937 and THP-1 (monocytic lineage), K-562 (erythroid lineage), and Molt-4 (T cell lineage). These susceptible cell lines greatly differed in the speed at which the CPE progressed. The progression of CPE was faster in relatively well-differentiated cell lines such as HL-60 and U-937, independently of the multiplicity of infection, than in less differentiated cell lines such as K-562, KG-1, and THP-1. Thus, for the same lineage, the speed at which CPE progressed became proportionally higher with subsequent differentiation stages. In the K-562 cell culture, CPE were not observed until at least 5 days postinfection (p.i.), while more than 80% of HL-60 cells were killed within 3 days p.i. There were no significant differences between infected HL-60 and K-562 cells in the efficiency of infection determined at 8 hr p.i. by the indirect immunofluorescent technique, the rate of virus growth, or the amount of viral capsid protein synthesized. This indicated that there were similar viral replication cycles in the two cell lines. These observations suggest that the killing function of the virus is expressed more slowly in K-562 cells than in HL-60 cells.

[Effect of non-histone chromatin proteins on the transcription of globin genes]. / Vliianie negistonovykh belkov khromatina na transkriptsiiu globinovykh genov.

Globin gene transcription was studied in vitro using chromatin of normal and Rauscher virus-transformed erythroid cells. For this purpose the reconstituted chromatin was obtained and used as a template for RNA synthesis. The RNA-transcript was then analysed for the presence of globin mRNA sequences. When chromatin was reconstituted using DNA from Rauscher-transformed erythroblasts and reticulocyte nonhistone proteins they enhanced transcription. Most of the globin gene activation was due to the nonhistone protein fraction eluted from hydroxylapatite in 0.05 M Na-phosphate and the RNA-transcript in this case contained the largest quantity of globin sequences.

Susceptibility to staphylococcal alpha-toxin of Friend virus-infected murine erythroblasts during differentiation.

Splenic erythroblasts obtained from BALB/c mice infected with the anemia strain of Friend virus were compared with "matured" cells and adult erythrocytes for their sensitivity to staphylococcal alpha-toxin. Matured cells were obtained by treating erythroblasts in culture with erythropoietin for 48 h. Sensitivity to staphylococcal alpha-toxin, measured both by release of 86Rb and by cell lysis, failed to demonstrate significant differences among the cell types. Since maturation of erythroblasts to matured cells or erythrocytes is associated with synthesis of band 3, hemoglobin, and spectrin and the loss of transferrin receptors, we conclude that none of these compounds serves as the specific receptor for staphylococcal alpha-toxin in BALB/c mice.

[Molecular cloning of provirus sequences of Rauscher leukemia virus from mouse erythroleukemia cell genome]. / Molekuliarnoe klonirovanie provirusnykh posledovatel'nostei leikoza Raushera iz genoma myshinykh éritroleikoznykh kletok.

Provirus from a component of Rauscher leukaemia virus (RLV) has been cloned. The provirus (the size of 5000 b. p.) contains two LTR sequences and shares expressed sequence homology with Mo-MuLV. Restriction analysis and determination of the LTR nucleotide sequence and of the site from 3'-end of proviral genome have shown the cloned provirus to be the SFEV component of RLV. LTR from this cloned provirus contains all sites necessary for transcription: CAAT and TATA sequences, "cap" site and polyadenylation signal. The LTR of the cloned provirus from SFEV component of RLV has been shown to function as a promoter in E. coli cells.

Transformation of erythroid cells by Rous sarcoma virus (RSV).

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive (ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42 degrees), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type (wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37 degrees) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60src kinase is involved in erythroid cell transformation. Since the src and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.